Switchable-hydrophilicity solvent liquid-liquid microextraction of non-steroidal anti-inflammatory drugs from biological fluids prior to HPLC-DAD determination.

Switchable-hydrophilicity solvent liquid-liquid microextraction was used prior to high-performance liquid chromatography with diode-array detector (HPLC-DAD) for the determination of four non-steroidal anti-inflammatory drugs [i.e., ketoprofen, etodolac, flurbiprofen and ibuprofen] in human urine, saliva and milk. Optimum extraction conditions were as follows: 500 µL switched-on N,N-dimethylcyclohexylamine as the extraction solvent, 9.5 mL of the aqueous phase, 500 µL 20 M sodium hydroxide as a switching-off trigger, and within 30 s extraction time. A portion of the final extract was directly injected into HPLC. Under optimized extraction and chromatographic conditions, limits of detection ranged between 0.04 and 0.18 µg mL-1 in all matrices analyzed. Good linearity with coefficients of determination (R2) ranging between 0.9955 and 0.9998, and percentage relative standard deviations (%RSD) of 0.9-7.7% were obtained. The proposed method was efficiently used for the extraction of the analytes in the biological fluids with percentage relative recoveries (%RR) ranging between 95.7 and 109.2%.

Isolation of an unknown metabolite of the non-steroidal anti-inflammatory drug etodolac and its identification as 5-hydroxy etodolac.

The non-steroidal anti-inflammatory drug etodolac is extensively metabolized in the liver. Renal elimination of etodolac mainly as glucuronide and its other phase I and phase II metabolites is the primary route of excretion. High-performance liquid chromatography assays of human urine after application of etodolac indicated the existence of a further monohydroxylated metabolite (metabolite X) that was identified as 5-hydroxy etodolac. For the identification, electrospray ionization mass spectrometry (ESI-MS) as well as 1H-nuclear magnetic resonance (1H-NMR) and 13C-NMR spectroscopy have been used.

Capillary electrochromatography-electrospray ionization mass spectrometry for the qualitative investigation of the drug etodolac and its metabolites in biological samples.

On-line coupling of capillary electrochromatography (CEC) with electrospray ionization (ESI) mass spectrometry (MS) was used for the qualitative investigation of the biotransformation of the non-steroidal anti-inflammatory drug etodolac. The coupling of this electro-driven separation technique with mass spectrometry allowed us to demonstrate the presence of different metabolites of etodolac extracted from human urine.

Separation and identification of etodolac and its urinary phase I metabolites using capillary electrochromatography and on-line capillary electrochromatography-electrospray ionisation mass spectrometry coupling.

Capillary high-performance liquid chromatography (capillary HPLC), pressure-assisted capillary electrochromatography (pCEC) and capillary electrochromatography (CEC) were performed in the same capillary packed with 5 microm octadecylsilica (C18) as stationary phase. These three separation modes were compared from the viewpoint of peak efficiency and separation selectivity in order to critically evaluate the advantages which CEC may offer compared to capillary HPLC for the solution of practical biomedical problems. The separation of the non-steroidal anti-inflammatory drug etodolac (ET, 1) and its phase I metabolites, 6-hydroxy etodolac (6-OH-ET, 2), 7-hydroxy etodolac (7-OH-ET, 3) and 8-(1'-hydroxyethyl) etodolac (8-OH-ET, 4) was selected as an example. Baseline separation of all compounds was achieved in different modes and conditions. The effect of pure electrophoretic separation mechanism on the overall separation selectivity observed in CEC has been shown. A high electroosmotic flow (EOF) was observed in C18 packed capillary even at pH 2.5 in various buffers. Furthermore, these separations were coupled on-line with electrospray ionisation mass spectrometry (ESI-MS) and the parent drug and its metabolites were identified in urine. For the coupling of CEC with ESI-MS a laboratory-made electrophoretic device was used in order to overcome some technical disadvantages of commercial instrumentation.

Effects of urinary metabolites of etodolac on diagnostic tests of bilirubin in urine.

Etodolac (CAS 41340-25-4) is a non-steroidal anti-inflammatory drug used clinically. The bilirubin levels in urine samples from patients treated with etodolac were determined with diagnostic tests (diazo and oxidized methods). The urine samples gave a positive reaction with a diazo method, but not with an oxidized method. The serum concentrations of bilirubin in the patients were also in the normal range. These results indicate that the positive reaction of the urine samples from patients treated with etodolac is false and caused by urinary metabolites of etodolac. To identify the false positive substance in the urine samples, the urinary metabolites of etodolac were extracted with ethyl acetate. The extract was injected into a high performance liquid chromatography (HPLC) column and the eluate was mixed with the diazo reagent. Three positive fractions were found. The retention times of the two metabolites in HPLC matched with those of the 6- and 7-hydroxylated metabolites of etodolac. In addition, authentic compounds of the 6- and 7-hydroxylated metabolites gave a positive reaction in the diazo methods. These results indicate that the 6- and 7-hydroxylated metabolites are mainly responsible for the false positive reactions of the urine sample from patients treated with etodolac.

Etodolac in equine urine and serum: determination by high-performance liquid chromatography with ultraviolet detection, confirmation, and metabolite identification by atmospheric pressure ionization mass spectrometry.

A high-performance liquid chromatographic method was used for the detection of etodolac in equine serum and urine. The method consisted of a one-step liquid-liquid extraction, separation on a reversed-phase (RP-18) column and detection using an ultraviolet detector. Additional confirmation methods included a HPLC coupled with an atmospheric pressure chemical ionization mass spectrometer (APCI-MS). Free (unbound) etodolac and its conjugates were present in the samples. Concentrations of the drug in the serum and urine samples collected from four standardbred mares after a single oral administration of Ultradol were determined. Maximum etodolac concentrations of 712, 716, 568, and 767 microg/mL in urine and 4.1, 3.6, 3.1, and 2.2 microg/mL in serum were observed. The peak concentrations of the drug were detected 2-10 h (urine) and 40 min-6 h (serum) after administration to four horses. The maximum detection time was 79 h in urine and 48 h in serum after the drug administration. The drug-elimination profiles for both urine and serum are presented and discussed. Method ruggedness and precision and stability studies of etodolac in serum and urine are presented. Three major metabolites were detected in the urine by liquid chromatography-APCI-MS. All three metabolites were identified as monohydroxylated etodolac.

Simultaneous determination of the phase II metabolites of the non steroidal anti-inflammatory drug etodolac in human urine.

Etodolac, an antirheumatic and analgetic drug, is metabolized in humans by hydroxylation and acyl glucuronidation to yield the corresponding 1-O-glucuronides. The acylglucuronides of etodolac and one of its hydroxylated metabolites were determined by HPLC using a RP18 column. In order to increase the lipophilicity of these metabolites the hydroxy groups were acetylated and the carboxylic functions were methylated. In a study with five human volunteers a stereoselective excretion of the acylglucuronides could be observed. The S-etodolac-glucuronide is mainly excreted during the first 6 hours after administration of 400 mg of etodolac whereas the elimination of the R-glucuronide predominates at longer periods of time. The S-glucuronide of 7-hydroxy-etodolac was preferenetively excreted during the period of time observed. An unknown metabolite of etodolac could be characterized by chemical and enzymatically hydrolysis and by MS and NMR.

Evaluation of the stereoselective metabolism of the chiral analgesic drug etodolac by high-performance liquid chromatography.

The enantiomers of the racemic analgesic drug etodolac have been resolved by fractional crystallization of the diastereomeric salts with optically active 1-phenylethylamine. A high-performance liquid chromatographic method to determine racemic etodolac (assay I) and its major metabolites (assay II) in urine using a conventional reverse-phase column is described. The determination of the enantiomeric ratios of etodolac and the two metabolites 7-hydroxyetodolac and 8-(1'-hydroxyethyl)etodolac was achieved using different protein-bonded chiral stationary phases. The urinary data for five volunteers are presented and show a marked stereoselectivity of the metabolism of etodolac in humans.

The stereoselective pharmacokinetics of etodolac in young and elderly subjects, and after cholecystectomy.

The pharmacokinetics of the enantiomers of etodolac were studied in six young subjects (ages 28 +/- 3.3 years), 6 nonarthritic elderly subjects (ages 73 +/- 6.0 years), and in three cholecystectomy patients after single oral doses of the racemate (200 mg). In all subjects, the plasma concentrations of R-etodolac, which is pharmacologically inactive, greatly exceeded those of the pharmacologically active S-enantiomer. Stereoselectivity was reflected in the pharmacokinetics, with R > S for maximum peak plasma concentration and area under the concentration versus time curve, and S > R for apparent oral clearance and apparent volume of distribution. On average, less than 25% of the dose of each enantiomer was excreted in the urine within 24 hours as alkali-labile conjugates; little or no unchanged drug was recovered. Bile constituted a minor route of elimination of etodolac as conjugated enantiomers. There were no significant differences in the pharmacokinetics of etodolac enantiomers between the young and elderly subjects. The results reflect the importance of considering stereoselectivity in evaluating the pharmacokinetics of etodolac.